A fundamental but unsolved question in neuroscience is how specific connections between neurons underlie information processing in the cortical circuits. Local circuits in the cerebral cortex consist of tens of thousands of neurons, each making thousands of connections. Perhaps the biggest reason we don't understand these circuits is that we have never been able to reconstruct their actual wiring diagrams. But if we had a partial or even complete wiring diagram, we would also need to know what each neuron in a circuit is doing: its physiology. In this proposal we plan to develop and apply new approaches for large-scale electron microscopy, towards the goal of mapping wiring diagrams of cortical circuits in a functional context. We will use two-photon calcium imaging to see the activity of neurons in a functioning local circuit. We will then use large-scale serial-section electron microscopy to trace circuits in the same piece of cortex. Recent advances in functional imaging and serial-section electron microscopy (EM) allow us to study this difficult problem, but before we can reap the benefits of these approaches, considerable technical work is necessary. Functional imaging with two-photon microscopy is a technically mature field, but approaches for large-scale serial-section EM are still in their infancy. We propose to apply the dual approach of functional imaging followed by high-resolution anatomical imaging-which we have already performed once in a large pilot project-with the goal of improving the technologies specifically for large-scale EM, correlated with functional studies. Our four-year goal is to create a high-throughput system for generating correlated structure/function data sets from the cortex. In particular, we will build a new EM imaging system, a second-generation Transmission EM Camera Array (TEMCA), that will allow us to capture very large three-dimensional data sets (300 to 500 micrometers on a side) in a week, rather than months. We propose to address one class of question: are there subnetworks within each local cortical circuit that process distinct information? But the approach is general and can be applied to a wide range of questions, including clinically relevant ones. Are neural connections disrupted near plaques in Alzheimer's disease? When stem cells incorporate into a circuit, do they form connections that play a functional role? For the first time, these questions should be within our reach. By developing high-throughput methods for large-scale imaging, we will begin to study neural circuits on their own terms: in all of their complexity and with data sets that are in many senses complete.